Prostasin is a serine protease which has been isolated from human seminal fluid. Prostasin localization in body fluids (seminal fluid and urine) of tissues has been reported, as revealed by radioimmunoassay (RIA) using an antiserum which was obtained with the use of purified prostasin as an antigen (Yu, J. X. et al., J. Biol. Chem., Vol. 269, pp. 18843-18848, 1994). Furthermore, full-length cDNA encoding human prostasin, the amino acid sequence thereof, and the structure thereof have already been reported (Yu, J. X. et al., J. Biol. Chem., Vol. 270, pp. 13483-13489, 1995).
Prostasin is present in the nature in the form of a complex formed by binding to an inhibitor protein or in a free form as in the case of other serine proteases. As prostasin inhibitor proteins, a prostasin-binding protein (PBP; PN-1) or hepatocyte growth factor activator inhibitor-1B (HAI-1) has been reported (Chen, L-M. et al., The Prostate, Vol. 59, pp. 1-12, 2004; and Fan B. et al., J. Biol. Chem., Vol. 280, pp. 34513-34520, 2005). A complex of prostasin with PBP has a molecular weight of approximately 82 kDa and is thermostable in the presence of SDS and a reducing agent. However, the presence of other inhibitors is predicted, in addition to the inhibitor proteins reported above. It is unknown that to what kinds of inhibitor proteins prostasin binds to form complexes in body fluids such as urine, seminal fluid, and blood.
The clinical significances of the measurement of prostasin, which have been reported, are: 1) the possible use thereof for diagnosis of hypertension; and 2) the possible use thereof as tumor marker for diagnosis, for example. Diagnosis of hypertension with the use of the measurement of prostasin has been suggested, since prostasin has effects on the activation of the epithelial Na channel (ENaC) which is important for regulation of renal tubular Na reabsorption and body fluid volume (Narikiyo, T. et al., J. Clin. Invest., Vol. 109, pp. 401-408, 2002; and Olivieri, O. et al., Hypertension, Vol. 46, pp. 683-688, 2005). Moreover, the use of prostasin as a tumor marker has been suggested, since serum prostasin levels in epithelial ovarian cancer patients are found to be significantly higher than those in normal groups (Mok, S. C. et al., J. Natl. Cancer Inst., Vol. 93, pp. 1458-1464, 2001). For measurement of prostasin, immunoassays (e.g., radioimmunoassay, immunoblotting, ELISA, and Western blotting) have been performed using an antiserum or a monoclonal antibody. which are obtained by immunization with the use of purified natural prostasin as an antigen (for example, Yu, J. X. et al., 1994; Narikiyo, T. et al., 2002; Mok, S. C. et al., 2001; and Chen, L-M. et al., Prostate, Vol. 59, pp. 1-12, 2004). In addition a peptide is designed based on the amino acid sequence of a known prostasin, an antiserum or a monoclonal antibody is prepared using the peptide as an antigen for immunization, and the antiserum or the monoclonal antibody is used in immunoassays (e.g., Western blotting) (for example, Olivieri, O. et al., 2005).
However, natural prostasin can not be stably obtained. So, it has been difficult to obtain an antibody using natural prostasin as an antigen. In addition, prostasin has 6 disulfide bonds, making it difficult to obtain a protein having a correct conformation when recombinantly expressed. Therefore, an antibody generated against the recombinant prostasin does not always react with natural prostasin. Furthermore, although an antibody against a partial peptide of prostasin is known to react with prostasin under denaturation conditions (for example, in Western blotting) (for example, Production Information of Mouse Anti-Human Prostasin Antibody (Catalog No. 612173, produced by BD Biosciences), such antibody could not be used in immunoassays under non-denaturation conditions.